Cells sequester and degrade their own intracellular contents by autophagy. This process is involved in a variety of biological pathways and disease processes, including host defense, cancer, and the starvation response. An intriguing new facet to macroautophagy has emerged in the form of its role in lipid breakdown and cholesterol efflux. Recent studies shed light on the consequences of this function, and point to the therapeutic potential of targeting the molecular components and regulators of autophagy for treatment of obesity, metabolic syndrome, and atherosclerosis.

Autophagy serves multiple purposes in the cell, removing damaged proteins and organelles, fighting intracellular pathogens, and mobilizing intracellular stores for energy during nutrient deprivation. Macroautophagy delivers cargo to the lysosome via a double-membraned vesicle, the autophagosome, and either sweeps up a sampling of cytosol regardless of what is in it, or selectively targets cargo like mitochondria, ribosomes, or protein aggregates (1).

A new twist in the autophagy story was introduced in 2009, with the discovery that the autophagy pathway targets lipid droplets for delivery to lysosomes in hepatocytes (2). Singh et al demonstrated that under normal conditions, autophagic delivery of lipids to lysosomes happens as just one component of an overall non-selective process. However, under conditions of increased lipolysis such as serum starvation, autophagy of lipid droplets becomes selective. They dubbed this lipid-selective autophagy "macrolipophagy". New studies have revealed that autophagy of lipid droplets plays a role in multiple disease processes, suggesting the potential of targeting autophagy-related molecules for treatment of conditions such as obesity and atherosclerosis.

Two sample-based research observed upregulated autophagy markers in subjects with obesity and Type II diabetes, suggesting a potential relationship between these disorders and enhanced autophagy, as well as macrolipophagy. A 2011 research comparing autophagy markers in adipose tissues from obese and non-obese tissue donors examined changes in the protein and mRNA of three autophagy genes, ATG5 and LC3A and B, in the omental and subcutaneous adipose tissue (3). LC3 is modified by ATG proteins to produce LC3-II, a component of the autophagosome membrane involved in cargo selection, while ATG5 is a marker for the isolation membrane (4-5). Obesity, especially in the omental tissue, was associated with more autophagosomes and autophagic flux, while insulin resistance correlated with higher expression of autophagy markers, indicating a relationship between these conditions and increased autophagy.

This is consistent with an earlier research by Ost et al., which studied adipocytes from obese subjects with Type II diabetes (6). Insulin was less able to activate mTORC1 in these cells, leading to overactivation of autophagy. This was determined by observation of high numbers of autophagosomes, as well as higher levels of LC3 in rapamycin- and chloroquine-treated cells from diabetic subjects compared to similarly treated cells from non-diabetic subjects. Higher numbers of LD were also observed in adipocytes from diabetic subjects, and colocalization of the LD marker perilipin with LC3 was also observed, suggesting that not only is general autophagy upregulated in these cells, but macrolipophagy occurs as well.

The relationship between lipid autophagy and obesity may be one of cause and effect, as Kaushik et al (2011) recently uncovered a new mechanism for macrolipophagy in regulating food intake (7). Agouti-related peptide (AgRP) neurons in the hypothalamus produce AgRP, a neuropeptide that antagonizes melanocortin receptors to promote food-seeking behavior. In this study, starvation responses in AgRP neurons triggered macrolipophagy, affecting food intake and fat mass in mice (see figure Macrolipophagy in AgRP neurons upregulates AgRP expression, increasing food intake).

While the presence of starvation-induced autophagy in the brain has been controversial (8-9), serum starvation induced upregulation in autophagy markers and autophagic flux in hypothalamic neurons, and food restriction did the same in mice, as measured via hypothalamic lysates and explants. Re-feeding decreased these markers, confirming the cause and effect relationship between nutrient deprivation and autophagy in this cell type. Intriguingly, impairing autophagy in AgRP neurons produced leaner mice with lower body weight, total fat mass, and white adipose tissue mass. The mice in this research ate less than controls when re-fed after a fast, and also displayed more physical activity. Offering a potential explanation for these behavioral and phenotypic changes, AgRP levels were lower in Atg7 knockout mice than in controls, and levels of the appetite-controlling hormone alpha-MSH were enhanced. However, the mechanistic link between autophagy and AgRP remained unclear.

Consistent with the high levels of free fatty acids produced during starvation and autophagy's link to the starvation response, autophagy in AgRP neurons seems to be enhanced by treatment with fatty acids. Oleic acid or palmitic acid treatment induced autophagic flux, colocalization of neutral lipids with lysosomal markers, and association of lipid droplets (LD) with autophagosome markers in serum-starved cells. This evidence strongly suggests that LD delivery to lysosomes happens through autophagy. Treating cells with serum from starved rats led to higher levels of triglycerides than in untreated cells, suggesting increased levels of fatty acid uptake during nutrient deprivation, and medial basal hypothalamus from fasted mice showed the same trend. Importantly, however, impairing autophagy by knockdown of the autophagy protein Atg5 elevated triglycerides and diminished fatty acid levels, implying involvement of autophagy in triglyceride degradation.

Finally, serum-starved cells made more than twice as much AgRP as fed cells, while adding fatty acids to fed cells caused as much upregulation of AgRP as starvation. However, inhibiting lysosomes or autophagy diminished upregulation of AgRP, strengthening the link between the lipophagic process and modulation of AgRP expression. The authors suggest that this link may point to treatments for disorders such as obesity. However, recent studies have also shown that sustained treatment with rapamycin, which activates autophagy by inhibition of mTOR, either induces weight loss and lower food intake, or staves off age-related weight gain (10-11). Given that hypothalamic mTOR signaling is known to suppress food intake in rats (12), these results indicate that tissue targeting may be key in extending Kaushik et al's findings to future treatments.

Macrolipophagy in foam cells may also be a key to reverse cholesterol transport (RCT), a process of cholesterol efflux and excretion that has been studied extensively for treating atherosclerosis. LD cholesterol release kicks off RCT, and recent research by Ouimet and colleagues describes specific induction of autophagy in lipid-loaded macrophages to deliver LD to the lysosome (13). Traditionally, neutral CE hydrolases are thought to be responsible for breakdown of cholesteryl esters (CE) stored in lipid droplets. However, the group showed LD colocalizing with lysosomes, implying that lysosomes may play a role as well. Indeed, blocking vesicle acidification while inhibiting CE hydrolase activity yielded a cumulative effect on increase in CE mass in cells, indicating involvement of both pathways.

A substantial portion of LD associated with the autophagosome marker LC3, suggesting that autophagy contributes significantly to cholesterol breakdown in lipid-loaded macrophages. Confirming this, blocking autophagosome-lysosome fusion reduced cholesterol efflux from macrophages to apoA-I. While lipid loading enhanced LC3-II levels and lysosomal inhibition diminished cholesterol efflux from lipid-loaded foam cells, this mechanism was not active in unloaded, LDL-treated macrophages, which retained their cholesterol efflux capacity in the presence of lysosomal inhibition. Therefore, higher intracellular cholesterol levels appear to be responsible for initiating the macrolipophagy pathway for efflux. Inhibition experiments showed that lysosomal acid lipase (LAL) is the enzyme responsible for hydrolyzing the CE during this process.

Observations in Atg5-/- macrophages confirmed the importance of autophagy in cholesterol efflux from lipid-loaded macrophage foam cells. All other steps of the process after lipid loading were normal: similar levels of cholesterol loading occurred compared to wild-type macrophages, and lipid droplet formation proceeded normally as well. However, efflux and CE hydrolysis were substantially diminished in the autophagy-impaired cells. Moreover, the LAL inhibitor reduced efflux in wild-type, but not autophagy-deficient macrophages.

The significance of lipophagy in cholesterol efflux is mixed depending on which acceptor is involved. While nearly all cholesterol efflux to ApoA-I depended on this process, macrolipophagy accounted for only about 30% of efflux to HDL, suggesting that efflux as a result of autophagy is unidirectional and involves ABCA-1. Moreover, lipophagy appears to play a significant role in RCT in vivo. Injection of wild-type lipid-loaded macrophages into mice led to more excretion of 3H-labeled cholesterol than did injection of autophagy-impaired cells, indicating that a robust macrolipophagic response is important for ridding the body of excess cholesterol. Ultimately, these findings may point to modulation of autophagy as a potential treatment for atherosclerosis.

As a newly emerging facet of autophagy, macrolipophagy has already shown diverse and important roles in a number of disease states. In addition to the findings in obesity, diabetes, and atherosclerosis, two recent experiments suggest a role for lipophagy in viral infection. Lipid autophagy appears to be triggered by the hepatitis C virus in infected cells, and higher levels of LC3-II relative to LC3-I correlate with less microvesicular steatosis in liver samples from infected subjects, indicating a potentially protective role for this process during HCV infection. Conversely, dengue virus regulates lipid metabolism via lipophagy to support its own replication within the cell (15-16). As new findings continue to emerge, it is likely that macrolipophagy will continue to shed light on a variety of processes important in biology and disease.